PH gradients controlled by electrolysis, and their use in isoelectric focusing

ABSTRACT

A specified proton concentration in a volume (80) is produced by passing a controlled electrophoresis current through an adjacent electrophoresis volume (28) between a working electrode (26) and a counter electrode (24). An array of such volumes with specified proton concentration is used to provide the pH gradient for isoelectric focusing.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/675,794 filed on Aug. 25, 2010, which is a National Phase of PCTPatent Application No. PCT/IL2008/001159 having International FilingDate of Aug. 26, 2008, which claims the benefit of priority of U.S.Provisional Patent Application Nos. 60/935,698, filed on Aug. 27, 2007and 61/039,257, filed on Mar. 25, 2008.

The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments, relates to the field ofmolecular interactions, and particularly to methods and devices usefulin producing local concentration of protons, proton concentrationgradients and desired proton concentration topographies in anenvironment (e.g., a solution, a gel, or the like) including anelectrolyte. Some embodiments of the invention also relate to the fieldof analyte separation and isoelectric focusing. Some embodiments of theinvention also relate to the field of data display.

Isoelectric focusing is an analytical technique for separating moleculesin an analyte sample by taking advantage of the differing ionicproperties of the molecules.

Isoelectric focusing is performed in a gel (usually of materials such aspolyarylamide polyacrylamide, starch or agarose) having an immobilizedproton concentration gradient, generally the proton concentrationgradient changing from higher to lower pH in a given direction.

The analyte is loaded onto some location on the gel. The charge of eachdifferent molecule changes in response to the ambient protonconcentration according to the acidity (pKa) of the various functionalgroups of the molecule.

An electric potential is applied parallel to the proton concentrationgradient between an isoelectric focusing anode and isoelectric focusingcathode. Molecules having a net positive charge migrate through the geltowards the anode while molecules having a net negative charge migratethrough the gel towards the cathode. The opposite, positively chargeions (cations) migrate towards the cathode and negatively charged ions(anions) migrate towards the anode.

As the molecules migrate, the ambient pH changes to reduce the netcharge on the molecule until the molecule reaches an isoelectric point(pI) where, due to the ambient pH, the net charge on the molecule iszero so that the molecule stops migrating due to the electric potential.If a molecule “overshoots” the isoelectric point, the molecule reversesdirection.

In such a way, isoelectric focusing focuses molecules having the same pIinto very narrow well-defined volumes of the gel.

Isoelectric focusing is exceptionally useful for the analysis ofproteins as proteins are characterized by having many functional groupsof different acidities.

Isoelectric focusing suffers from a number of disadvantages. To havesufficient resolution, it is often necessary to have a number ofdifferent gels having different proton concentration gradients spanningdifferent ranges of proton concentration gradients, increasing costs andcreating a logistical problem. Automated manipulation of fragile gels isdifficult to implement. Gels having immobilized proton concentrationgradients are generally expensive and may suffer from batch to batchreproducibility. Analysis of some analytes may suffer from a sievingeffect as large proteins may have difficulty migrating through pores inthe gels. Isoelectric focusing may be slow due to the slow migration ofthe analyte molecules.

It would be advantageous to be able to perform isoelectric focusing withfewer disadvantages of the methods known in the art.

SUMMARY OF THE INVENTION

The invention relates, in some embodiments, to the field of molecularinteractions, and particularly to proton concentration topographies,methods and devices useful in producing specified local concentrationsof protons and specified proton concentration topographies in anenvironment including an electrolyte that, in some embodiments, aremutable and may be changed as desired or that are temporally variable.

Some embodiments of the present invention provide for isoelectricfocusing that is devoid of at least some of the disadvantages of themethods known in the art.

Some embodiments of the present invention provide for the display ofdata.

In some embodiments, the invention provides a device comprising aplurality of independently controllable cells, arranged for example in aone- or two-dimensional array, and an environment including anelectrolyte. Each cell is configured to independently produce aspecified proton concentration in an associated volume of theenvironment. When the device is activated, a user can use the cells toproduce a desired one- or two-dimensional proton concentrationtopography in the environment, where each volume of the environment hasa specified proton concentration that is substantially not dependent onthe proton concentration in adjacent volumes.

In some embodiments, the proton concentration topography is mutable. Insome such embodiments, a user may choose to change the protonconcentration in some or all of the volumes, and consequently the protonconcentration topography, by controlling the cells. In some embodiments,the user may choose to change the proton concentration in some or all ofthe volumes as a function of time, and consequently the protonconcentration topography as a function of time.

Thus, according to an aspect of some embodiments of the invention thereis provided proton concentration topography, comprising: a) anenvironment including an electrolyte, the environment divided into aplurality of neighboring discrete volumes; b) between any twoneighboring volumes, an interface volume; and c) each volume having aspecified proton concentration.

Depending on the embodiment, the volumes are arranged in one dimension,for example constituting a one-dimensional array of volumes, in twodimensions, for example constituting a two-dimensional array of volumes,or arranged in three dimensions, constituting a three-dimensional array.

According to some embodiments, in a given direction through theenvironment the rate of change of proton concentration betweenneighboring volumes is substantially monotonous.

According to some embodiments, in a given direction through theenvironment, the rate of change of proton concentration betweenneighboring volumes varies.

According to some embodiments, the proton concentration topography iscontrollably mutable. According to some embodiments, the protonconcentration topography is controllably mutable as a function of time.

According to an aspect of some embodiments of the invention there isprovided a method of producing a specified proton concentrationtopography in an environment including an electrolyte, comprising:

-   -   a) providing a plurality of independently controllable cells,        each cell configured to produce a specified proton concentration        in an associated volume of an environment including an        electrolyte, for example by electrolysis of components of the        environment    -   b) contacting an environment including an electrolyte with the        plurality of cells so as to divide the environment into a        plurality of neighboring discrete volumes, each volume        associated with a cell;    -   c) specifying a desired proton concentration topography; and    -   d) activating each cell of the plurality of cells, so as to        produce a specified proton concentration in each volume        associated with a cell in the environment,        wherein the specified proton concentrations produced in each        volume collectively constitute the specified proton        concentration topography.

According to an aspect of some embodiments of the invention there isprovided a device for the production of a proton concentrationtopography in an environment including an electrolyte, comprising:

-   -   a) a plurality of independently controllable cells, each cell        configured to produce a specified proton concentration in an        associated volume of an environment including an electrolyte,        for example by electrolysis of a component of the environment,        substantially independently of other the cells; and    -   b) a container functionally associated with the plurality of        cells configured to contain an environment including an        electrolyte, the container configured to allow production of the        specified proton concentration by a cell in an associated volume        of an environment contained in the container.

According to an aspect of some embodiments of the invention there isprovided a method for producing a specified proton concentration in anenvironment including an electrolyte, comprising:

-   -   a) providing an environment including an electrolyte;    -   b) contacting a working electrode and a counter electrode with        the environment so that:        -   a first side of the working electrode faces the counter            electrode to define an electrolysis volume of the            environment between the working electrode and the counter            electrode, and        -   a second side of the working electrode faces a second volume            of the environment;    -   c) specifying a desired proton concentration;    -   d) passing a current between the working electrode and the        counter electrode so as to electrolyze components of the        environment to generate electrolysis products (e.g., ions) in        the electrolysis volume; and    -   e) transferring at least some of the electrolysis products to        the second volume, thereby producing a proton concentration in        the second volume of the environment        wherein the current is such that the proton concentration        produced in the second volume is the specified proton        concentration. According to some embodiments, the working        electrode is permeable to the passage of electrolysis products        therethrough (e.g., is made of mesh) and the transferring        includes allowing electrolysis products generated in the        electrolysis volume to pass through the working electrode into        the second volume, thereby producing the proton concentration in        the second volume.

According to an aspect of some embodiments of the invention there isprovided a device for producing a specified proton concentration in anenvironment including an electrolyte, comprising:

-   -   a) a working electrode; and    -   b) a counter electrode,    -   the working electrode and the counter electrode arranged so        that:        -   a first side of the working electrode faces the counter            electrode to define an electrolysis volume between the            working electrode and the counter electrode, and        -   a second side of the working electrode faces a volume of a            container, the container configured to contain an            environment including an electrolyte,            wherein the electrolysis volume is in fluid communication            with the volume of the container. According to some            embodiments, the working electrode is permeable to the            passage of electrolysis products therethrough (e.g., is made            of mesh) to allow electrolysis products generated in the            electrolysis volume to pass through the working electrode            into the second volume.

According to an aspect of some embodiments of the invention, there isprovided a method of analyzing an analyte using isoelectric focusing,comprising:

-   -   a) placing an analyte suitable for analysis by isoelectric        focusing in an environment including an electrolyte;    -   b) producing a non-immobilized proton concentration topography        in the environment;    -   c) applying a potential difference to the environment; and    -   d) observing locations of components of the analyte in the        environment in relation to the proton concentration topography        and the potential difference        thereby analyzing the analyte using isoelectric focusing.        According to some embodiments, the non-immobilized proton        concentration topography is mutable.

According to an aspect of some embodiments of the invention, there isprovided a device useful in implementing the method of isoelectricfocusing.

According to an aspect of some embodiments of the invention, there isprovided a method of displaying data, comprising:

-   -   a) providing a plurality of independently controllable cells,        each cell configured to produce a specified proton concentration        in an associated volume of an environment including an        electrolyte, for example by electrolysis;    -   b) contacting an environment with the plurality of cells so as        to divide the environment into a plurality of neighboring        discrete volumes, each of the plurality of discrete volumes        associated with a cell, the environment including:        -   an electrolyte, and        -   a pH sensitive indicator having an appearance that is            dependent on the proton concentration in the environment;            and    -   c) activating each cell of the plurality of cells, so as to        produce a specified proton concentration in each associated        volume of the environment        wherein the specified proton concentrations produced in each        volume gives a specified appearance to the pH indicator; and        where the collective appearance of the pH indicator in the        volumes is an image that constitutes a display of the data.

According to an aspect of some embodiments of the invention, there isprovided a device useful in implementing the method of displaying data.

Unless otherwise defined, technical and/or scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In case of conflict, the patentspecification, including definitions, will control. The materials,methods, and examples disclosed herein are illustrative only and are notintended to be necessarily limiting.

As used herein, the terms “comprising”, “including” and “having” orgrammatical variants thereof are to be taken as specifying the statedfeatures, integers, steps or components but do not preclude the additionof one or more additional features, integers, steps, components orgroups thereof. This term encompasses the terms “consisting of” and“consisting essentially of”.

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

Unless the context dictates otherwise, the terms “generate” and“produce” or grammatical variants thereof are to be consideredsynonymous.

Herein, the terms “array of cells” and “electrode array” are in someinstances used interchangeably.

Herein, the terms “analyte” and “material” are in some instances for thesame concept in a different context. The term “analyte” generally refersto a material in an analytical context (e.g., analysis of the amount ofthe material present in a mixture) while the more general “material”refers, for example, to a material in a preparatory context.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying figures. Thedescription, together with the figures, makes apparent how embodimentsof the invention may be practiced to a person having ordinary skill inthe art. The figures are for the purpose of illustrative discussion ofembodiments of the invention and no attempt is made to show structuraldetails of an embodiment in more detail than is necessary for afundamental understanding of the invention. For the sake of clarity,most objects depicted in the figures are not to scale.

In the Figures:

FIG. 1A schematically depicts various one-dimensional protonconcentration topographies;

FIG. 1B schematically depicts various two-dimensional protonconcentration topographies;

FIG. 1C schematically depicts, in perspective, devices for producingproton concentration topographies;

FIG. 2A schematically depicts, in side cross section, a device forproducing a specified proton concentration;

FIG. 2B is a graph qualitatively showing the concentration of productsof electrolysis in the device of FIG. 2A;

FIGS. 3A-3C schematically depict a device for producing aone-dimensional proton concentration topography suitable for use inisoelectric focusing;

FIGS. 4A-4C schematically depict a device for producing aone-dimensional proton concentration topography suitable for use inisoelectric focusing;

FIGS. 5A-5C schematically depict a device for producing aone-dimensional proton concentration topography configured for use inisoelectric focusing;

FIGS. 6A-6B schematically depict a device for producing aone-dimensional proton concentration topography configured for use inisoelectric focusing;

FIGS. 7A and 7B are schematic top views of devices for producingone-dimensional proton concentration topographies, the devices actuallyconstructed and used for implementing the teachings of the invention;

FIGS. 8A-8B are graphs showing proton concentrations produced using thedevice depicted in FIG. 7A;

FIG. 8C is a graphs showing a proton concentration topography producedusing the device depicted in FIG. 7B;

FIGS. 9A-9B schematically depict an embodiment of a device for producinga one-dimensional proton concentration topography configured for use inisoelectric focusing; and

FIGS. 10A-10F schematically depict an embodiment of a method of thepresent invention for separating analytes in a mixture.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

The invention, in some embodiments, is of proton concentrationtopographies as well as methods and devices for producing protonconcentration topographies in an environment (e.g., a solution, gel orthe like) including an electrolyte, generally environments having a low,negligible or non-existent buffering capacity. In some embodiments, theproton concentration topographies are non-immobilized, that is to say,are produced on-demand, when desired and as needed. In some embodiments,the produced proton concentration topographies are mutable, that is tosay may be controllably changed at will or as a function of time. Someembodiments also relate to proton concentrations topographies in fluidssuch as liquids (as opposed, for example, to gels) that are useful, forexample for isoelectric focusing and for purifying analytes. Someembodiments relate to or are useful for implementing isoelectricfocusing. Some embodiments relate to or are useful for implementingisoelectric focusing in fluids. Some embodiments relate to or are usefulfor the display of data.

The principles, uses and implementations of the teachings of theinvention may be better understood with reference to the accompanyingdescription and figures. Upon perusal of the description and figurespresent herein, one skilled in the art is able to implement theteachings of the invention without undue effort or experimentation. Inthe figures, like reference numerals refer to like parts throughout.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth herein. The invention can be implemented withother embodiments and can be practiced or carried out in various ways.It is also understood that the phraseology and terminology employedherein is for descriptive purpose and should not be regarded aslimiting.

As noted above, some embodiments of the invention relate to protonconcentrations in an environment including an electrolyte. Such anenvironment allows the electrolytic generation of chemical entities,such as protons and hydroxyl anions that influence the protonconcentration in the environment. Typical such environments includeaqueous solutions and gels known in the art of isoelectric focusing andelectrophoresis.

In the art, proton concentration gradients are known. For example, whena current is passed between two electrodes immersed in a non-bufferedenvironment including an electrolyte, a monotonous proton concentrationgradient is produced, a high proton concentration close to the anodethat continuously and monotonously drops to a low proton concentrationclose to the cathode. Such proton concentration gradients arenon-immobilized as these are produced only when desired. Such protonconcentration gradients are also mutable as changing the electricalcurrent passing between the electrodes changes the gradient.

In contrast, prior art isoelectric focusing methods are based on the useof non-mutable proton concentration gradients immobilized in a non-fluidenvironment such as a gel.

Some embodiments of the invention relate to proton concentrationtopographies in an environment. By proton concentration topography ismeant the distribution of proton concentrations in the environment andas such includes the term “proton concentration gradient”. However,while the term “proton concentration gradient” evokes a linear,continuous and monotonously changing proton concentration as a functionof location, the term “proton concentration topography” has a broadermeaning and includes distributions of proton concentrations inone-dimension (e.g., a line, curve, outline of a geometric figures suchas a circle), in two-dimensions (e.g., a planar surface, a curvedsurface, a concave surface, a convex surface) and in three-dimensions.The term “proton concentration topography” includes topographies wherein a given direction through the environment the rate of change ofproton concentration is not continuous and/or not monotonous.

In some embodiment, a proton concentration is mutable and can be changedin a controlled fashion. Thus, in embodiments a proton concentrationtopography may have a desired arbitrary shape or pattern that, inembodiments, may be changed in a controllable fashion. In someembodiments, there is provided temporal and spatial control of protonconcentration in an environment to allow generation of a controllableand adjustable proton concentration topographies, allowing generation ofdynamic and complex proton concentration topographies that can bechanged at will. In some embodiments, the control is with the use of anelectronic or digital controller.

In some embodiments of the invention, a proton concentration topographyis produced in a fluid environment, for example a liquid such as anaqueous solution. This allows implementation of isoelectric focusing andother methods using an ad hoc proton concentration topography producedin the fluid environment. Further, the use of a fluid environment inwhich to perform analyte separation allows for implementation of methodsfor preparing pure materials and not just for analysis of the mixture.An analyte mixture is separated using, for example, isoelectricfocusing. A desired component, e.g. a single protein, is easily isolatedfrom other components by extraction of the fluid including the component(e.g., with a pipette). This is much simpler and allows isolation ofmuch greater amounts of material than the currently required excision ofa volume of a gel. Thus, according to an aspect of some embodiments, ofthe present invention there is provided a method of isolating a material(e.g., purifying or concentrating the material) from a mixture ofmaterials (e.g., a polypeptide such as a protein or peptide from amixture of polypeptides) comprising: a) performing isoelectric focusingof a mixture of materials including a desired material in a fluidenvironment; and b) subsequent to the isoelectric focusing, removing thefraction of the fluid environment including the desired material,thereby isolating the desired material from the mixture of materials.

Proton Concentration Topographies

In some embodiments the invention provides a proton concentrationtopography, comprising: a) an environment including an electrolyte, theenvironment divided into a plurality of neighboring discrete volumes; b)between any two neighboring volumes, an interface volume; and c) eachvolume having a specified proton concentration.

In some embodiments, the proton concentration topographies of theinvention are discrete and not continuous. In some embodiments, the factthat the proton concentration topography is made up of a plurality ofneighboring discrete volumes, each volume having a specified protonconcentration means that each volume can be considered as a “pH-xel” (inanalogy to a pixel for two-dimensional images).

In some embodiments, the volumes are arranged in one dimension, forexample constituting a one-dimensional array of volumes, such as a line,curve or outline of a geometric figure such as a circle.

In some embodiments, the volumes are arranged in two dimensions, forexample constituting a two-dimensional array of volumes, such as ahexagonal array where each non-edge volume is surrounded by sixequidistant neighboring volumes or a square array where each non-edgevolume is surrounded by four neighboring volumes arranged as a cross.

In some embodiments, the volumes are arranged in three dimensions,constituting a three-dimensional array.

In some embodiments, the movement of ions between two neighboringvolumes is inhibited, for example by the interposition of a barrierimpermeable to ions between two volumes. In some such embodiments, theinterface volume is occupied by the barrier. Some such embodiments areexceptionally useful, for proton concentration topographies used for thedisplay of data where it may be advantageous to have a sharpdifferentiation between the volumes (pH-xels).

In some embodiments, the movement of ions between two neighboringvolumes is substantially uninhibited and d) each interface volume has aproton concentration related to the proton concentrations of theneighboring volumes defining the interface volume. Some such embodimentsare exceptionally useful for proton concentration topographies used forimplementation of isoelectric focusing where there is a need for analytemolecules to travel through the proton concentration topography until anisoelectric point is reached.

In some embodiments, in a given direction through the environment, therate of change of proton concentration between neighboring volumes issubstantially monotonous, for example is linear according to a molar ora pH scale.

In some embodiments, in a given direction through the environment, therate of change of proton concentration between neighboring volumesvaries, for example there are groups of neighboring volumes where therate of change of proton concentration from volume to volume is muchgreater or much lesser than other groups, there are discontinuities orthere are reversals in the rate of change.

In FIG. 1A are schematically depicted various embodiments of protonconcentration topographies.

One dimensional topographies i-vi are all produced in a elongated vesseldivided into ten neighboring discrete volumes where the protonconcentration (in pH) of each volume and each interface volume isdepicted with the help of graphs showing pH as a function of volumenumber.

In proton concentration topography i, the proton concentrationmonotonously increases at a constant (in pH units) at a high rate from avery low proton concentration in volume 1 to a very high concentrationin volume 10. It is seen that in each volume, the pH is well-definedwhile in the interface volumes the proton concentration is a gradientrelated to the proton concentrations of the neighboring volumes definingthe interface volume.

In proton concentration topography ii, the proton concentrationmonotonously increases at a constant (in pH units) at a low rate from acertain proton concentration in volume 1 to a somewhat higher protonconcentration in volume 10.

In proton concentration topography iii, the proton concentrationmonotonously increases from volume 1 to volume 10 at a varying rate.From volume 1 to volume 4 the rate of increase in proton concentrationis very high, while from volume 4 to volume 10 the rate of increase inproton concentration is relatively low.

In proton concentration topography iv, the proton concentrationmonotonously increases from volume 1 to volume 10 at a varying rate.From volume 1 to volume 2 the rate of increase in proton concentrationis very high, from volume 3 to volume 8 the rate of increase in protonconcentration is relatively low and from volume 8 to 10 the rate ofincrease in proton concentration is very high.

In proton concentration topography v, the proton concentrationmonotonously increases from volume 1 to volume 6 at a constant rate (inunits of pH) and then monotonously decreases from volume 6 to 10 at aconstant rate (in units of pH).

In proton concentration topography vi, the movement of ions between twoneighboring volumes is inhibited, for example by the placement ofimpermeable barriers between any two volumes. It is seen that in such anembodiment, the interface volume is, in fact, occupied by theimpermeable barrier and has no significant proton concentration.

Proton concentration topographies vii and viii depicted in FIG. 1B aretwo-dimensional proton concentration topographies schematically depictedfrom above. In proton topographies vi and vii, interface volumes aredepicted in grey. The proton concentration of each volume is written inunits of pH in the center of the volume while the proton concentrationof the interface volumes is defined by the neighboring volumes definingthe interface volume.

Proton concentration topography vii comprises sixteen discreteneighboring volumes, arranged in a 4 volume by 4 volume square arraywhere each non-edge volume is surrounded by four neighboring volumesarranged as a cross.

Proton concentration topography vii comprises 25 discrete neighboringvolumes, arranged in a hexagonal array where each non-edge volume issurrounded by six equidistant neighboring volumes.

In some embodiments of the invention, a proton concentration topographyis non-immobilized and is produced in an environment only when desired.

In some embodiments, the proton concentration topography is controllablymutable. In some embodiments, the proton concentration in at least onevolume, preferably a plurality of volumes may be controllably changed sothat the proton concentration topography is controllably mutable.

In some embodiments, the proton concentration topography is controllablymutable as a function of time. In some embodiments, the protonconcentration in at least one volume, preferably a plurality of volumes,may be controllably changed as a function of time so that the protonconcentration topography is controllably mutable as a function of time.

Methods for Producing a Proton Concentration Gradient

Any suitable method may be used to produce a proton concentrationgradient. That said, some embodiments provide a method that may be usedto produce a proton concentration gradient.

Some embodiments of the invention provide a method of producing aspecified proton concentration topography in an environment including anelectrolyte, comprising: a) providing a plurality of independentlycontrollable cells, each cell configured to produce a specified protonconcentration in an associated volume of an environment including anelectrolyte, for example by electrolysis; b) contacting an environmentincluding an electrolyte with the plurality of cells so as to divide theenvironment into a plurality of neighboring discrete volumes, eachvolume associated with a respective cell; c) specifying a desired protonconcentration topography; and d) activating each cell of the pluralityof cells, so as to produce a specified proton concentration in eachvolume associated with a cell of the environment, wherein the specifiedproton concentrations generated in each volume collectively constitutethe specified proton concentration topography.

In some embodiments, e) subsequent to d, a desired proton concentrationtopography that is different than a previously defined protonconcentration topography is specified; and f) where necessary, a cell ofthe plurality of cells is activated, to produce a different protonconcentration in the associated volume of the environment, therebychanging the proton concentration topography to be the newly specifiedproton concentration topography.

In some embodiments, the method further comprises changing (stepwise orcontinuously) a proton concentration in at least one associated volumeas a function of time, thereby changing the proton concentrationtopography as a function of time.

In some embodiments, the volumes are arranged in one dimension, forexample constituting a one-dimensional array of volumes, such as a line,curve or outline of a geometric figure such as a circle.

In some embodiments, the volumes are arranged in two dimensions, forexample constituting a two-dimensional array of volumes, such as ahexagonal array where each non-edge volume is surrounded by sixequidistant neighboring volumes or a square array where each non-edgevolume is surrounded by four neighboring volumes arranged as a cross.

In some embodiments, the volumes are arranged in three dimensions,constituting a three-dimensional array.

In some embodiments, the movement of ions between two the neighboringvolumes is inhibited, for example by the interposition of physicalbarriers, e.g., sheets of impermeable material such as polyethylene orglass or other barriers impermeable to ions between two volumes. Somesuch embodiments are exceptionally useful when the method is used forthe display of data where it may be advantageous to have a cleardifferentiation between the volumes.

In some embodiments, the movement of ions between two neighboringvolumes is substantially uninhibited, for example the environment is asubstantially homogenous (in the absence of different protonconcentrations), for example a solution or a gel devoid of animmobilized proton concentration gradient. Some such embodiments areexceptionally useful when the method is used for implementation ofisoelectric focusing where there is a need for analyte molecules totravel through the produced proton concentration topography until anisoelectric point is reached.

Devices for Producing a Proton Concentration Gradient

Any suitable device may be used to implement the method for producing aproton concentration gradient of the invention. That said, someembodiments of the invention provide a device that may be used toimplement a method for producing a proton concentration gradient.

Some embodiments of the invention provide a device for the production ofa proton concentration topography in an environment including anelectrolyte, comprising: a) a plurality of independently controllablecells, each cell configured to produce a specified proton concentrationin an associated volume of an environment including an electrolyte, forexample by electrolysis of a component of the environment, substantiallyindependently of the other cells; and b) a container functionallyassociated with the plurality of cells, the container configured tocontain an environment including an electrolyte and configured to allowproduction of the specified proton concentration by the cell in a volumeof the environment contained in the container, associated with the cell.In some embodiments, a plurality of cells is at least 2 cells, in someembodiments at least 5 cells, in some embodiments at least 10 cells, andin some embodiments at least 12 cells.

As noted above, in some embodiments, each volume associated with a cellmay be considered as an independently controllable “pH-xel”, where arespective cell may be used to produce a specified proton concentrationin the associated volume. Such configuration of a device allows adesired proton concentration topography to be produced by the device,including a topography having arbitrary features, by changing the protonconcentration in one or more of the associated volumes.

In some embodiments, the device further comprises an environmentincluding an electrolyte contained within the container.

In some embodiments, the device is configured to allow substantiallyuninhibited movement of ions between two neighboring volumes. Some suchembodiments are exceptionally useful when the device is used forimplementation of isoelectric focusing where there is a need for analytemolecules to travel through the produced proton concentration topographyuntil an isoelectric point is reached.

In some embodiments, the container has dimensions substantially largerthan the dimensions of the volumes associated with the cells. In somesuch embodiments, a single large container is functionally associatedwith a plurality of cells, and the volumes of environment associatedwith the cells are physically continuous and defined by the protonconcentration produced therein by an associated cell.

In some embodiments, the plurality of cells is arranged so that anenvironment held in the container is physically divided into a pluralityof discrete neighboring volumes, each such volume being a volumeassociated with a cell, as described above. In such embodiments, thecontainer may be considered a collection of subcontainers, each suchsubcontainer associated with a respective cell and in some embodimentsat least partially defining the associated volume in which therespective cell produces a specified proton concentration. In some suchembodiments, a subcontainer has dimensions of the order of a cell.

In some embodiments, the device further comprises barriers locatedbetween neighboring volumes and at least partially definingsubcontainers, the barriers configured to inhibit the movement of ionsbetween two neighboring volumes. Some such embodiments are exceptionallyuseful when the device is used for the display of data where it may beadvantageous to have a sharp differentiation between the volumes. Insuch devices, neighboring volumes are generally close together and thebarrier to ion movement is generally thin in relation to the size of thecells, for example, being less than 30%, less than 20% or even less than10% of the distance between the centers of the two volumes which thebarrier separates.

In some embodiments, the cells are arranged so that the associatedvolumes are arranged in one dimension, for example constituting aone-dimensional array of volumes, such as a line, curve or outline of ageometric figure such as a circle.

In some embodiments, the cells are arranged so that the associatedvolumes are arranged in two dimensions, for example constituting atwo-dimensional array of volumes, such as a hexagonal array where eachnon-edge volume is surrounded by six equidistant neighboring volumes ora square array where each non-edge volume is surrounded by fourneighboring volumes arranged as a cross.

In some embodiments, the cells are arranged so that the associatedvolumes are arranged in three dimensions, constituting athree-dimensional array.

In some embodiments, each cell is configured to change the specifiedproton concentration in the associated volume, in some embodimentssubstantially independently of the other cells. In some suchembodiments, such configuration allows a desired proton concentrationtopography to be produced, for example a topography having arbitraryfeatures, by changing the proton concentration in one or more of theassociated volumes.

In some embodiments, each cell is configured to change the specifiedproton concentration in the associated volume as a function of time, insome embodiments substantially independently of other the cells. In somesuch embodiments, such configuration allows a produced protonconcentration topography to be temporally variable.

In some embodiments, a device is configured to allow passage of anindividually controllable electrical current through each of the cellswhen an environment including an electrolyte fills the container. Insome such embodiments, such configuration allows a specified protonconcentration topography to be produced.

In some embodiments, a device further comprises a controller configuredto allow independent variation of the magnitude of an electrical currentpassing through each of the cells. In some embodiments, the controlleris configured to vary the electrical input as a function of time,allowing the proton concentration topography to be changed as a functionof time.

In some embodiments, the device further comprises a proton concentrationsensor functionally associated with each of the cells, the protonconcentration sensor configured to determine the value of a protonconcentration in the volume associated with a cell and to report thedetermined value to a controller; and the controller is configured tochange a magnitude of an electrical current passing through a cell inresponse to the reported value. In some embodiments, the devicecomprises a proton concentration sensor is used as a component of afeedback circuit to provide better control of a cell.

In some embodiments, the device further comprises a controller inputcomponent functionally associated with the controller, configured toallow a user to input a desired value for each of the cells, the desiredvalue influencing the magnitude of an electrical current passing throughthe cell.

A number of embodiments of devices for producing a proton concentrationgradient are depicted in FIG. 1C.

Device 10 comprises ten independently controllable cells 12 arranged ina line to constitute a linear area, each cell 12 configured to produce aspecified proton concentration in an associated volume of an environmentincluding an electrolyte held in container 14. The volumes associatedwith each cell 12 are the volume of environment held in container 14proximal to that cell 12. As there are no barriers or other impedimentsbetween the associated volumes, device 10 is configured to allowsubstantially uninhibited movement of ions between neighboring volumes.

Device 10 comprises a controller 16 that is functionally associated witheach of cells 12 and is used to control the proton concentrationproduced in the associated volume of each cell 12 by controlling themagnitude of an electric current passing through each cell 12.Controller 16 includes a controller input component allowing a user toinput a desired proton concentration produced in an associated volume byeach cell in order to specify a desired proton concentration topography.Device 10 may be used to produce many different proton concentrationtopographies, including proton topographies i, ii, iii, iv and vdepicted in FIG. 1A.

Device 18 depicted in FIG. 1C is similar to device 10. However,container 14 is divided into ten subcontainers 14′ by impermeablebarriers 20.

In embodiments where container 14 is filled with a fluid environmentincluding an electrolyte so that the level of fluid is lower than theheight of barriers 20, the environment held in container 14 is dividedinto a plurality of physically discrete neighboring volumes where ionmovement between neighboring volumes is inhibited. In such embodiments,each volume associated with a cell 12 is physically defined by asubcontainer 14′. In such embodiments, device 18 may be used to producemany different proton concentration topographies, including protonconcentration topography vi depicted in FIG. 1A.

In some embodiments, container 14 is filled with a fluid environmentincluding an electrolyte so that the level of fluid is higher than theheight of barriers 20. In such embodiments, each volume associated witha cell 12 is only partially physically defined by a subcontainer 14′. Insuch embodiments, device 18 may be used to produce many different protonconcentration topographies, including proton topographies i, ii, iii, ivand v depicted in FIG. 1A.

Device 22 depicted in FIG. 1C comprises sixteen independentlycontrollable cells 12 arranged in a four cell by four cell square array,each cell 12 configured to produce a specified proton concentration inan associated volume of an environment including an electrolyte held incontainer 14. The volumes associated with each cell 12 are the volume ofenvironment held in container 14 proximal to that cell 12. As there areno barriers or other impediments between the volumes, device 10 isconfigured to allow substantially uninhibited movement of ions betweenneighboring volumes. Device 22 may be used to produce many differentproton concentration topographies, including proton concentrationtopography vii depicted in FIG. 1B.

Method for Producing a Specified Proton Concentration in an Environment

Implementation of embodiments of the invention for producing a protonconcentration topography as described above are contingent on producinga specified proton concentration in an environment, for example in avolume of an environment associated with a cell as described above.

Any suitable method for producing a specified proton concentration in anenvironment may be used. That said, some embodiments of the inventionprovide a method for producing a specified proton concentration in anenvironment including an electrolyte, comprising: a) providing anenvironment including an electrolyte; b) contacting a working electrodeand a counter electrode with the environment so that: a first side ofthe working electrode faces the counter electrode to define aelectrolysis volume of the environment between the working electrode andthe counter electrode, and a second side of the working electrode facesa second volume of the environment; c) specifying a desired protonconcentration; d) passing a current between the working electrode andthe counter electrode so as to electrolyze components of the environmentto generate electrolysis products in the electrolysis volume; and e)transferring at least some electrolysis products to the second volume,thereby producing a proton concentration in the second volume of theenvironment, wherein the current is such that the proton concentrationproduced in the second volume is the specified proton concentration.

In some embodiments, the working electrode is permeable to the passageof the electrolysis products therethrough (e.g., is made of mesh) andthe transferring of electrolysis products includes allowing theelectrolysis products generated in the electrolysis volume to passthrough the working electrode into the second volume, thereby producingthe proton concentration in the second volume.

In some embodiments, the working electrode is an anode and the counterelectrode is a cathode.

In some embodiments, the working electrode is a cathode and the counterelectrode is an anode.

In some embodiments the method further comprises: f) monitoring(continuously or periodically) the proton concentration produced in thesecond volume of the environment; and g) if necessary, adjusting thecurrent passing between the working electrode and the counter electrodeso as to maintain the proton concentration produced in the second volumeas the specified proton concentration.

In some embodiments, the method further comprises: subsequent to d,specifying a desired proton concentration different than a previouslyspecified proton concentration; and passing a current between theworking electrode and the counter electrode, thereby producing a protonconcentration in the second volume of the environment, wherein thecurrent is such that the proton concentration produced in the secondvolume is the different specified proton concentration.

In some embodiments, the method further comprises: changing (stepwise orcontinuously) the current as a function time, thereby changing theproton concentration produced in the second volume as a function oftime.

Device for Producing a Specified Proton Concentration in an Environment

Any suitable device for producing a specified proton concentration in anenvironment may be used in implementing the teachings of the invention.

That said, some embodiments provide a device that may be used toimplement a method for producing a specified proton concentration in anenvironment including an electrolyte, comprising: a) a workingelectrode; and b) a counter electrode, the working electrode and thecounter electrode arranged so that: a first side of the workingelectrode faces the counter electrode to define a electrolysis volumebetween the working electrode and the counter electrode, and a secondside of the working electrode faces a second volume of a container, thecontainer configured to contain an environment including an electrolyte,wherein the electrolysis volume is in fluid communication with thesecond volume of the container.

In some embodiments, the working electrode is permeable to the passageof electrolysis products therethrough (e.g., is made of mesh) to allowelectrolysis products generated in the electrolysis volume to passthrough the working electrode into the second volume.

In some embodiments, the device further comprises an environmentincluding an electrolyte contained within the container, filling theelectrolysis volume and contacting the working electrode and the counterelectrode.

In some embodiments, the container has dimensions of the order of thecounter electrode and of the second volume, and the container is atleast partially physically defined by a barrier impermeable to thepassage of ions.

In some embodiments, the container has dimensions substantially largerthan the dimensions of the second volume.

In some embodiments, the device is configured to allow establishment ofan electrical circuit between the working electrode and the counterelectrode when an environment including an electrolyte fills thecontainer and the electrolysis volume.

In some embodiments, the device further comprises a controllerconfigured to allow variation of the magnitude of an electrical currentpassing through an established electrical circuit.

In some embodiments, the device further comprises a proton concentrationsensor functionally associated with the cell and with the controller,the proton concentration sensor configured to determine the value of aproton concentration in the second volume and to report the value of theproton concentration to the controller, and the controller is furtherconfigured to change a magnitude of an electrical current passingthrough an established electrical circuit in response to the reportedvalue. In some embodiments, comprises a proton concentration sensor isused as a component of a feedback circuit to provide better control of acell.

In some embodiments, the device further comprises a controller inputcomponent functionally associated with the controller, configured toallow a user to input a desired value that influences the magnitude ofan electrical current passing through an established electrical circuit.

In some embodiments, the controller is configured to vary the electricalcurrent passing through an established electrical circuit as a functionof time, allowing a proton concentration generated in the second volumeto be changed as a function of time.

In FIG. 2A, a device for producing a specified proton concentration inan environment, cell 12 is schematically depicted in side cross-section.Cell 12 comprises a counter electrode 24, a working electrode 26 (a meshpermeable to the passage of ions), an electrolysis volume 28 (the volumebetween working electrode 26 and counter electrode 24), a second volume30 on the opposite side of working electrode 26 and a protonconcentration sensor 32 in second volume 30, all contained within thevolume defined by container 14. Electrodes 24 and 26 are functionallyassociated with controller 16, which is configured to control themagnitude of a current passing between electrodes 24 and 26 when anenvironment including an electrolyte is contained within container 14and in contact with electrodes 24 and 26.

For use, an environment including an electrolyte is added to container14 filling electrolysis volume 28 and second volume 30 and establishingan electrical circuit including electrodes 24 and 26. Controller 16passes an electrical current through the established circuit betweenelectrodes 24 and 26. Electrolysis occurs in electrolysis volume 28,electrolyzing water to generate electrolysis products such as protonsand hydroxyl anions. The protons migrate to the cathode while thehydroxyl anions migrate to the anode, changing the proton concentrationin the vicinity of the electrodes.

Ions in the vicinity of working electrode 26 pass through workingelectrode into second volume 30, changing the proton concentration insecond volume 30 to be substantially equal to that near workingelectrode 26.

Proton concentration sensor 32 determines the value of a protonconcentration in second volume 30 and reports the value of the protonconcentration to controller 16 which then, if necessary, changes themagnitude of the electrical current in response to the reported value inorder to produce a desired proton concentration in second volume 30, asinput to controller 16 by a user through the controller input componentof controller 16.

In FIG. 2B is shown a graph qualitatively depicting the concentration ofprotons and hydroxyl anions in device 22 of FIG. 2A when activated sothat working electrode 26 is an anode and counter electrode 24 is acathode. It is seen that the proton concentration monotonously decreasesthrough electrolysis volume 28 from a maximum near counter electrode 24.In the proximity of working electrode 26 as well as in second volume 30there is a high concentration of hydroxyl anions and consequently a lowconcentration of protons. It is seen that the concentration of hydroxylanions in all of second volume 30 is the same and substantially the sameas the concentration near working electrode 26.

Methods and Devices for Isoelectric Focusing

As discussed above, isoelectric focusing is based on providing asuitable proton concentration topography, for example, a topographywhere the concentration of protons changes monotonously in a givendirection, and application of a potential difference parallel to thedirection. In the art, isoelectric focusing is performed in protonconcentration gradients immobilized in gels.

The teachings of the invention may be applied to perform isoelectricfocusing, where the proton concentration topography is not-immobilized.

Some embodiments of the invention provide a method of analyzing ananalyte using isoelectric focusing, comprising: a) placing an analytesuitable for analysis by isoelectric focusing in an environmentincluding an electrolyte; b) producing a non-immobilized protonconcentration topography in the environment; c) applying an electricpotential difference to the environment; and d) observing locations ofcomponents of the analyte in the environment in relation to the protonconcentration topography and the electric potential difference, therebyanalyzing the analyte using isoelectric focusing.

Observing the locations of the components is by any suitable method andgenerally depends on the nature of the analytes. For example, in someembodiments, fluorescent analytes (e.g., proteins) are observed with thehelp of a fluorescence detector.

In some embodiments, the environment is a fluid. This is in contrast toknown methods of isoelectric focusing where the environment in which theproton concentration topography needed for isoelectric focusing is agel. The use of a fluid allows for quicker isoelectric focusing as thecomponents of the analytes migrate to the isoelectric point morequickly, is cheaper as no expensive and sensitive gels need to bemanipulated, and allows high-throughput analysis as a single device maybe used to serially analyze analytes automatically: after analysis of ananalyte is completed, the fluid is drained, the device washed and freshfluid introduced.

In some embodiments, the proton concentration topography is mutable. Insome embodiments, the method further comprises: e) subsequent to d,changing the mutable proton concentration topography to a secondnon-immobilized proton concentration topography; and f) observing thelocation of components of the analyte in the environment in relation tothe second proton concentration topography and the electric potential.Such embodiments allow performance of multiple analyses of the samesample. For example, a first proton concentration topography covering abroad range of proton concentrations is used to provide a roughindication of the isoelectric points of the components of the analyte(i.e., at “low resolution”). Subsequent proton concentrationtopographies are produced, each such topography including a limitedrange of proton concentrations spanning the proton concentrationsnecessary to determine the isoelectric point of a specific component ofthe analyte (i.e., at “high resolution”). In such a way, the isoelectricpoint of each component of an analyte may be determined with greataccuracy.

In some embodiments, the method further comprises: subsequent to d,while observing, intermittently or continuously, locations of componentsof the analyte in the environment, changing the proton concentrationtopography as a function of time. Such embodiments are useful for“scanning” an unknown analyte to determine a limited range ofisoelectric points before performing a more exacting analysis.

In some embodiments, devices for isoelectric focusing according to theinvention are devices for producing a proton concentration topography,for example such as described above, but provided with an anode and acathode for applying the isoelectric focusing field. When necessary, ionconcentrations and the like may be determined using commerciallyavailable components, for example based on ion sensitive field-effecttransistors technology.

As noted above, some embodiments of the invention are directed tomethods and devices for generating a proton concentration topography foruse in isoelectric focusing and related analyte separation methods.

An embodiment of a device configured producing a proton concentrationtopography and useful for isoelectric focusing in accordance with theteachings of the invention, device 36, is depicted in FIGS. 3A(perspective), 3B (side view) and 3C (top view).

Casing 34, substantially a non-conductive rectangular box of transparentpolycarbonate with an open top, about 5 cm long, 0.5 cm wide, 0.5 cmdeep contains other components of device 36. The volume defined bycasing 34 is considered to be a container 14.

On one side of casing 34 is found a substantially standardelectrophoresis assembly comprising an isoelectric focusing anode 38, anisoelectric focusing cathode 40 and an electrophoresis gel 42 (e.g.,neutral uncharged polyacrylamide hydrogel available from Bio-Rad Haifa,Ltd., Haifa, Israel). By functionally associating anode 38 and cathode40 with a standard electrophoresis power source, electrophoresis may beperformed in the usual way in gel 42.

One face of gel 42 contacts a wall of casing 34. Contacting the opposingface of gel 42 is a membrane 44 (e.g., a hydrophilic polyvinylidenefluoride (PVDF) membrane with 5 micrometer pores available, for example,as Durapore (SVLP04700) from Millipore, Inc. Billerica, Mass., USA) thatphysically supports gel 42 and allows substantially free passage of ionsand water molecules between gel 42 and the rest of the volume ofcontainer 14.

Container 14 contains components of an array of a plurality (ten)independently controllable cells, similar to cell 12 depicted in FIG.2A. Each cell of the array of cells is configured to produce a specifiedproton concentration in an associated volume of gel 42. Takencollectively, cells of the array of cells are configured for producing aone-dimensional (linear) proton concentration topography in gel 42 inaccordance with aspects of the invention.

The array of cells includes a single counter electrode 24 (platinumsheet) opposing ten individually controllable working electrodes 26 a-26j (platinum mesh, made of 0.1 mm thick wires with 0.1 mm gaps), eachworking electrode 26 separated from neighboring working electrodes 26 byan electrode separator 46 a-46 i (0.1 mm glass walls). Workingelectrodes 26 a-26 j are placed about 0.5 mm from membrane 44 and about2 mm from counter electrode 24 by.

The volume defined between working electrodes 26 and counter electrode24 is an electrolysis volume 28.

The volume defined by a working electrode 26, one or two borderingelectrode separators 46 and membrane 44 is a proton reservoir volume 48a-48 j, on the bottom of which is embedded a proton concentration sensor32 a-32 j (e.g., Orion 9863BN, Thermo Fisher Scientific Inc., Waltham,Mass., USA) and a magnetic stirrer 50 a-50 j.

For use, electrolysis volume 28 and proton reservoir volumes 48 a-48 jare filled with an electrolyte solution (e.g., 0.1 M Na₂SO₄ in water).

Isoelectric focusing anode 38, isoelectric focusing cathode 40, counterelectrode 24, working electrodes 26, proton concentration sensors 32 andstirrers 50 are all functionally associated with a controller 16 (e.g.,an appropriately configured microprocessor or digital computer withnecessary peripheral accessories).

Stirrers 50 and proton concentration sensors 32 are activated.

Controller 16 activates each cell so that a selected current, typicallybetween 0 and about 1 mA cm⁻² (depending on the desired protonconcentration) is independently established between counter electrode 24and each working electrode 26 a-26 j so that a voltage of between 0 andabout 5V exists between counter electrode 24 and each working electrode26 a-26 j.

As the system stabilizes, a proton concentration gradient (substantiallyas depicted in FIG. 2B) is generated in the environment including anelectrolyte held in electrolysis volume 28 between counter electrode 24and each working electrodes 26 a-26 j. As discussed with reference toFIG. 2B, in each proton reservoir volume 48 a-48 j, a single stable anduniform proton concentration is maintained due to the small volume of aproton reservoir volume 48 as well as due to the action of stirrers 50.The proton concentration in each proton reservoir volume 48 influencesthe proton concentration in gel 42 through membrane 44 so that theproton concentration in a given proton reservoir volume 48 is the samein a volume of gel 42 immediately adjacent to that proton reservoirvolume 48.

With reference to proton concentration sensors 32, the current passingbetween counter electrode 24 and each working electrode 26 a-26 j ischanged until a desired proton concentration (as specified by controller16) is produced and detected in an associated proton reservoir volume48. As the current passing between counter electrode 24 and each workingelectrode 26 a-26 j is independently controllable, the protonconcentration in each proton reservoir volume 48 and consequently in theadjacent volume of gel 42 is separately controllable. In such a way, adesired specified proton concentration topography is produced in gel 42,where each cell produces a specified proton concentration in a volume ofgel 42 associated with that cell.

A mixture of one or more analytes for separation by isoelectric focusingis loaded onto gel 42 and a potential (e.g., 700 V) is supplied betweenisoelectric focusing anode 38 and isoelectric focusing cathode 40. Theproton concentration topography inside gel 42 causes separation of theindividual analytes in the mixture according to the isoelectric points.The locations of the individual analytes are observed and analyzed inthe usual way. Once the mixture of analytes has been separated andanalyzed at a first proton concentration topography, the potential onworking electrodes 26 is optionally changed to generate a differentproton concentration topography in order to have a different isoelectricseparation which is also observed and analyzed in the usual way.

For example, in an embodiments a first proton concentration topographyis specified to have a proton concentration having pH 5.0 in the volumeof gel 42 associated with working electrode 26 a, the protonconcentration decreasing monotonously and linearly to a protonconcentration having a pH 8.0 in the volume of gel 42 associated withworking electrode 26 j, while a second proton concentration topographyis specified to have a proton concentration having pH 5.2 in the volumeof gel 42 associated with working electrode 26 a, the protonconcentration decreasing monotonously and linearly to a protonconcentration having a pH 5.9 in the volume of gel 42 associated withworking electrode 26 j.

For example, in an embodiments a first proton concentration topographyis specified to have a proton concentration having pH 5.0 in the volumeof gel 42 associated with working electrode 26 a, the protonconcentration decreasing monotonously and linearly to a protonconcentration having a pH 6.0 in the volume of gel 42 associated withworking electrode 26 j, while a second proton concentration topographyis specified to have a proton concentration having pH 5.8 in the volumeof gel 42 associated with working electrode 26 a, the protonconcentration decreasing monotonously and linearly to a protonconcentration having a pH 7.0 in the volume of gel 42 associated withworking electrode 26 j.

In the specific examples described hereinabove, in the direction throughgel 42 from proximity with working electrode 26 a to proximity withworking electrode 26 j, the proton concentration monotonously decreases.As is clear to one skilled in the art, the change in protonconcentration in a direction through gel 42 may be substantially anydesired function, monotonous or not.

An additional embodiment of a device configured for producing a protonconcentration topography and useful for isoelectric focusing inaccordance with the teachings of the invention, a device 52, is depictedin FIGS. 4A (perspective view), 4B (side view) and 4C (top view).

Device 52 substantially resembles device 36 but includes a number ofnotable differences. One difference is that there is not one counterelectrode 24 that functions opposite a plurality of working electrodes26 a-26 j but rather a plurality of independent counter electrodes 24a-24 j, each functionally associated with a respective working electrode26 a-26 j. An additional difference is that electrode separators 46 a-46i separate neighboring counter electrodes 24 a-24 j as well asneighboring working electrodes 26 a-26 j, thereby defining a pluralityof physically discrete electrolysis volumes 28 a-28 j. Additionally,proton concentration sensors 32 are embedded inside a gel 42 on a wallof a casing 34 opposite a respective counter electrode 24 so as tomeasure the actual proton concentration of a volume associated with aworking electrode 26 inside gel 42. Further, device 52 is substantiallydevoid of proton reservoir volumes 48. Rather, the distance betweenworking electrodes 26 and a membrane 44 is very small, approximatelyonly 0.1 mm.

An additional embodiment of a device configured for producing a protonconcentration topography and useful for isoelectric focusing inaccordance with the teachings of the invention, a device 54, is depictedin FIGS. 5A (perspective view), 5B (side view) and 5C (top view).

Device 54 substantially resembles devices 36 and 52 but includes anumber of notable differences, including that device 54 is configured sothat proton concentration sensors 32 are isolated from the influence ofthe electric field generated by isoelectric focusing anode 38 andcathode 40. Each proton reservoir volume 48 is divided into two parts,as seen in FIG. 5B, a first part 48′ (in FIG. 5B, 48 a′ is depicted) anda second part 48″ (in FIG. 5B, 48 a″ is depicted).

A first part of a proton reservoir volume 48, such as 48 a′, is definedby a working electrode 26, one or two bordering electrode separators 46and membrane 44 so that protons and water molecules can passsubstantially uninhibited between first part 48′ and gel 42.

A second part of a proton reservoir volume 48, such as 48 a″, is definedby a working electrode 26 and one or two bordering electrode separators46. Second part 48″ is separated from first part 48′ and from gel 42 byan insulating partition 56, for example of polycarbonate, so thatmovement of protons between second part 48″ and gel 42 is inhibited.Proton concentration sensors 32 (in FIG. 5B, 32 a is depicted) arecontained within second part 48″ of a volume 48. In such a way, protonconcentration sensors 32 are isolated from the electric field betweenisoelectric focusing anode 38 and cathode 40.

An additional embodiment of a device configured for producing a protonconcentration topography and useful for isoelectric focusing inaccordance with the teachings of the invention, device 58, is depictedin FIGS. 6A (perspective view) and 6B (side view).

Device 58 substantially resembles devices 36, 52 and 54 but includes anumber of notable differences. Like in device 36, device 58 includesproton reservoir volume 48 a-48 j, defined by a working electrode 26,one or two bordering electrode separators 46 and a proton-permeablemembrane 44 a intimately associated with gel 42. Like in device 54,proton concentration sensors 32 are positioned on a wall of a casing 34opposite a counter electrode 24, separated from direct contact with agel 42 by a proton-permeable membrane 44 b. However, unlike in device54, in device 58 proton concentration sensors 32 are not embedded in gel42 but rather contact a surface thereof.

An additional embodiment of a device configured for producing a protonconcentration topography and useful for isoelectric focusing inaccordance with the teachings of the invention, device 66, is depictedin FIGS. 9A (perspective view) and 9B (top detailed view). Device 66substantially resembles devices 36, 52, 54 and 58 but includes a numberof notable differences.

Device 66 includes an array of five independently controllable cells 12a-12 e substantially similar to cell 12 depicted in FIG. 2A. Each cellof the array of cells is configured to produce a specified protonconcentration in an associated volume of an environment including anelectrolyte held in proton concentration topography channel 72. Takencollectively, cells 12 of the array of cells are configured forproducing a one-dimensional (linear) proton concentration topography inchannel 72.

Device 66 is provided with a large anode bath 68 in which isoelectricfocusing anode 38 is immersed and a large cathode bath 70 in whichisoelectric focusing cathode 40 is immersed.

In device 66, components of each cell 12 are isolated from protonconcentration topography channel 72 in separate volumes of container 14to protect the components from damage from the electric field generatedbetween anode 38 and cathode 40. Counter electrode 24 and workingelectrode 26 are isolated in a volume (which also includes anelectrolysis volume 28 and a proton reservoir volume 48), the volume influid communication with proton concentration topography channel 72through a narrow conduit that constitutes a portion of a respectiveproton reservoir volume 48. The volume of an environment including anelectrolyte that is found in proton concentration topography channel 72in proximity to the opening of each conduit together with protonreservoir volume 48 comprises the volume associated with each cell 12 inwhich a specified proton concentration is produced. In protonconcentration topography channel 72, between any two such volumes arefound interface volumes having a proton concentration (and in somecases, a proton concentration gradient) related to the protonconcentrations of the two neighboring volumes defining the interfacevolume.

Each proton concentration sensor 32 is isolated in a respective protonconcentration sensor volume 74 from proton concentration topographychannel 72 in fluid communication with proton concentration topographychannel 72 through a narrow conduit. In some embodiments (not depicted)pumps (e.g., peristaltic pumps) or other suitable device provide fluidcommunication between a proton concentration sensor volume 74 and arespective proton reservoir volume 48 to ensure that the proper protonconcentration is measured.

In devices 36, 52, 54 and 58, the environment including an electrolytein which a specified proton concentration topography is producedcomprises the electrolyte solution and gel 42. In devices 66, theenvironment including an electrolyte in which a specified protonconcentration topography is produced comprises the electrolyte solutionheld in proton concentration topography channel 72.

In devices 36, 52, 54, 58 and 66, the container configured to containthe environment including the electrolyte comprises container 14,including the portion in which a gel 42 is held or the protonconcentration topography channel 72 of device 66.

In devices 36, 52, 54 and 58, the individual independently controllablecells configured to produce a specified proton concentration in anassociated volume of an environment including an electrolyte comprisecounter electrode 24, electrolysis volume 28, a working electrode 26, aproton concentration sensor 32 and for devices 36, 54, 58 and 66, aproton reservoir volume 48.

In device 36, the electrolysis volumes of each cell are not physicallyseparated and there is a single counter electrode 24 for all workingelectrodes 26 a-26 j. However, in embodiments where a potential isapplied between an isoelectric focusing anode 38 and an isoelectricfocusing cathode 40, it is often advantageous that each workingelectrode 26 is provided with a dedicated counter electrode 24 to reduceinterference between neighboring cells. Consequently, in devices 36, 52,54, 58 and 66, each cell includes an electrolysis volume 28 between aworking electrode 26 and a respective counter electrode 24.

In devices 36, 54 and 58, the volume of environment associated with acell in which a specified proton concentration is produced includes aproton reservoir volume 48 as well as the volumes of membrane(s) 44 andgel 42 located proximally to a respective proton reservoir volume 48. Indevices 52, the volume of environment associated with a cell in which aspecified proton concentration is produced includes a the volumes ofmembrane 44 and gel 42 located proximally to a respective workingelectrode 26.

In devices 36, 52, 54, 58 and 66, proton concentration sensors 32measure proton concentrations produced by a cell (e.g., 12) and reportthe measured concentration to controller 16. This allows monitoring ofthe actually produced proton concentrations and, if necessary,adjustment of the current passing between a working electrode 26 and acounter electrode 24 to maintain the actually produced protonconcentration as a specified proton concentration.

As described above, when a device 36, 52, 54, 58 and 66 is operating toproduce a proton concentration topography, gel 42 or environment held inchannel 72 is divided into a plurality of discrete volumes characterizedby a specified proton concentration, each volume associated with and inproximity to a specific working electrode 26 and in the case of devices36, 54, 58 and 66 a respective proton reservoir volume 48. In gel 42 andin channel 72, between any two such associated volumes (and close toelectrode separators 46) are interface volumes having a protonconcentration (and in some cases, a proton concentration gradient)related to the proton concentrations of the two neighboring volumesdefining the interface volume.

In some embodiments, a device of the invention such as device 36, 52,54, 58 or 66 is configured so that a produced proton concentrationtopography in a gel 42 or proton concentration topography channel 72 ismutable and a proton concentration topography may be changed as desiredand/or varied with time.

In some such embodiments, a controller 16 is provided with a userinterface that allows a user to provide instructions specifying adesired proton concentration produced by each cell or to specify adesired proton concentration topography, as needed. Upon receipt of userinstructions, controller 16 changes (or maintains) the current passingbetween one or more counter electrodes 24 and respective workingelectrodes 26 so as to produce the desired proton concentrationtopography.

In some such embodiments, controller 16 is provided with a timer andinstructions as how to change a produced proton concentration topographyas a function of time. With reference to the timer, controller 16changes (or maintains) the current passing between one or more counterelectrodes 24 and respective working electrodes 26 in accordance withthe instructions so as to change the produced proton concentrationtopography as a function of time. The change in current may be such thatthe change in proton concentration produced by a cell changes in astep-wise fashion (that is to say, is allowed to stabilize at a certainvalue and is maintained at that value for some time) or continuously.

In some embodiments, such as device 36, each proton reservoir volume 48is provided with a dedicated stirrer 50. In some embodiments, a protonreservoir volume is devoid of an actual stirrer and a uniform protonconcentration is achieved by diffusion of protons inside the protonreservoir volume.

In some embodiments operation of a device such as a device 36, 52, 54,58 or 66 may lead to the generation of heat, especially when a currentpasses between isoelectric focusing anode 38 and isoelectric focusingcathode 40. In some such embodiments, a device is provided with acooling system, for instance comprising components to cool theenvironment including an electrolyte. In a typical example, a devicesuch as device 66 depicted in FIG. 9 is provided with cooling elementsin the walls of anode bath 68 and cathode bath 70.

In the embodiments discussed above, isoelectric focusing is performed ina portion of the environment that is gel 42 or in channel 72 in which aproton concentration topography is produced that is non-immobilized andis mutable. Some advantages include the ability to use cheaper gels withgreater reproducibility and the possibility, discussed herein, toanalyze the same sample of analyte using a number of different protonconcentration topographies.

An embodiment demonstrating the utility of a mutable protonconcentration gradient when performing isoelectric focusing is discussedwith reference to FIGS. 10A-10F. In the embodiment, it is desired toresolve two proteins of interest having a similar electrophoreticmobilities but different isoelectric points (e.g., at pH 5.5 and at pH6.5), the two proteins in a mixture together with three other proteinshaving different electrophoretic mobilities and unknown isoelectricpoints. Each of FIGS. 10A-10F schematically depicts the location of thefive proteins of the mixture (each depicted as a horizontal strip) ingel 42, a standard electrophoresis/isoelectric focusing gel, in which aproton concentration topography is produced and across which anelectrical field is applied between isoelectric focusing anode 38 andisoelectric focusing cathode 40. A characteristic of the embodiment isthat electrophoretic separation and isoelectric resolution are performedon parallel axes, and not perpendicular axes as known in the art.

A uniform proton concentration (pH 7) is produced throughout gel 42 andthe mixture of proteins is loaded onto gel 42 in the usual way. Apotential is applied between anode 38 and cathode 40.

In FIG. 10A, the individual proteins move at different rates move as aresult of electrophoresis.

In FIG. 10B, the individual proteins have reached the maximalelectrophoretic resolution with the “fastest” protein proximal to anode38. It is seen that the two proteins of interest are close together.

In FIG. 10C, a complex proton concentration topography is produced tofurther resolve the proteins of interest. In a central region 76, thevolume of gel 42 in which the two proteins of interest are found, theproton concentration monotonously decreases from pH 2 (on the side ofanode 38) to pH 10 (on the side of cathode 40). From the end of centralregion 76 having pH 2 towards cathode 40, a cathode region 78 (a volumeof gel 42 which includes two “unwanted” proteins) the protonconcentration decreases monotonously from pH 2 to pH 10 proximal tocathode 40. From the end of central region 76 having pH 10 towards anode38, an anode region 80 (a volume of gel 42 which includes an “unwanted”protein) the proton concentration increases monotonously from pH 10 topH 2 proximal to anode 38.

In FIG. 10D, the three “unwanted” proteins in cathode region 78 andanode region 80 move each to a respective isoelectric point near theedges of gel 42 while the two proteins of interest in central region 76are increasingly spatially resolved due to isoelectric focusingresulting from the proton concentration topography described withreference to FIG. 10C and the potential applied between anode 38 andcathode 40.

In FIG. 10E, central region 76 is lengthened, increasing the spatialresolution of the two proteins of interest. Cathode region 78 and Anoderegion 80 are made smaller, forcing the three “unwanted” proteins closertogether,

In FIG. 10F, the two proteins of interest in central region 76 arespatially well-resolved.

Thus, using a mutable proton concentration topography, the presentinvention provides a method of analyzing an analyte using isoelectricfocusing, as described above, where the second non-immobilized protonconcentration topography is chosen so that components of interest arespatially resolved to a greater extent than in the precedingnon-immobilized proton concentration topography.

In some embodiments of the invention, isoelectric focusing is performedin an environment that is a fluid such as a liquid, e.g., an electrolytesolution, for example in channel 72 of device 66 or in some suchenvironments gel 42 is removed and replaced with electrolyte solution.Some advantages in performing isoelectric focusing in a liquid is lowerprice, simpler replacement of the environment between analyses and nosieving effects that occur when large analyte molecules pass throughchannels inside gels. As discussed above, the use of a fluid environmentalso allows simple implementation of preparatory methods based onseparation methods such as isoelectric focusing.

In the embodiments discussed above, the movement of ions between twoneighboring volumes of the environment in gel 42 is substantiallyuninhibited, a configuration that is suitable for many uses, for exampleisoelectric focusing.

In some embodiments, the movement of ions between two neighboringvolumes is inhibited. For example, one such embodiment where ionmovement between neighboring volumes is substantially inhibited issimilar to device 36 depicted in FIG. 3 where gel 42 is replaced with aninsulating glass plate. In such an embodiment, the volume of theenvironment associated with an individual cell in which a specifiedproton concentration is produced includes only a respective protonreservoir volume 48. Such embodiments are useful, for example for thedisplay of data.

For use, proton reservoir volumes 48 and electrolysis volume 28 arefilled with an environment including an electrolyte and a pH sensitiveindicator having an appearance that is dependent on the protonconcentration in the environment, e.g. Yamada Universal pH indicator(see for example, Foster S F and Gruntfest in J. Chem. Educ. 1937, 14,274). The individual cells are activated to produce specified protonconcentrations in the respective proton reservoir volume 48. Theindicator in proton reservoir volumes 48 adopts a color that isdependent on the produced proton concentration. The proton concentrationin each proton reservoir volumes 48 is specified so that when thecollective appearance of proton reservoir volumes 48 (that is of theproduced proton concentration topography as made apparent by the pHindicator in the environment) constitutes a display of data for examplean image.

As is clear to one skilled in the art, the display of data using adevice similar to device 36 as described herein above having aone-dimensional array of cells producing a one-dimensional protonconcentration topography is limited. Analogous devices having atwo-dimensional array of cells to produce a two-dimensional protonconcentration topography have greater utility, especially for thedisplay of actual images.

Although an embodiment of the method and the device for displaying datawere described where the movement of ions between two neighboringvolumes is inhibited, in some embodiments for the display of datamovement of ions between two neighboring volumes is substantiallyuninhibited.

Embodiments of the present invention are useful in fields other thanisoelectric focusing and display of data, for example in microfluidicsand lab-on-chip applications.

The teachings of the present invention may be implemented by a personhaving ordinary skill in the art upon perusal of the specification andfigures using usual techniques and methods, for example as known in thefield of microelectronics and microfluidics.

EXPERIMENTAL

Device for Producing a Specified Proton Concentration

An embodiment of a device for the production of a proton concentrationin a liquid environment including an electrolyte, device 60 was made andused in accordance with the teachings of the invention to produce aspecified proton concentration. Device 60 is schematically depicted inFIG. 7A in top view.

Casing 34 of device 60 is a block of polymethyl(methacrylate) (Perspex®)4 cm long (y dimension), 3 cm wide (x dimension) and 2 cm high (zdimension) in which a container 14 was hollowed to accommodate a cell 12for producing a specified proton concentration in an environmentincluding an electrolyte, cell 12 being 1 cm long (y dimension), 0.6 cmwide (x dimension) and 1 cm deep (z dimension). Counter electrode 24 of0.1 mm thick platinum mesh was placed along a wall of cell 12. Workingelectrode 26 was placed inside container 14, in parallel to and spaced 2mm from counter electrode 24. Proton concentration sensor 32 (Orion9863BN, Thermo Fisher Scientific Inc., Waltham, Mass., USA) was placedon the wall of cell 12 opposite counter electrode 24, inside volume 30.

Container 14 was filled with an electrolyte solution of 0.1 M Na₂SO₄ inwater. Proton concentration sensor 32 was connected to a suitabledisplay device to indicate what proton concentration was measured bythat sensor in volume 30.

Device 60 was used produce a specified proton concentration in anenvironment held in volume 30. Counter electrode 24 and workingelectrode 26 were functionally associated with a variable power supplyto establish an electrical circuit. The variable power supply was usedto pass a current of between about 0 and about 1 mA cm⁻² at a potentialof between about 0 and about 5V through the electrical circuit includingthe electrodes. The current passing through the circuit led tohydrolysis of water and the generation of protons and hydroxyl anions inthe volume of electrolysis chamber 28. The concentration of protonsincreased in proximity of the cathode while the concentration of protonsdecreased in proximity of the anode. Since working electrode 26 was madeof mesh and therefore permeable to ions, generated ions from thevicinity of a working electrode 26 passed through working electrode 26into the electrolyte solution held in volume 30.

The magnitude of the current passing through the circuit was adjustedwith reference to the readings of proton concentration sensor 32 until aproton concentration corresponding to a pH of 4.7 was produced in theelectrolyte solution held in volume 30, see FIG. 8A.

The magnitude of the current passing through the circuit wassubsequently adjusted with reference to the readings of protonconcentration sensor 32 until a proton concentration corresponding to apH of 9 was produced in the electrolyte solution held in volume 30, seeFIG. 8B.

Device for Producing a Specified Proton Concentration Topography

An embodiment of a device for the production of a proton concentrationtopography in a liquid environment, device 62 was made and used inaccordance with the teachings of the invention to produce a specifiedproton concentration topography. Device 62 is schematically depicted inFIG. 7B in top view.

Casing 34 of device 60 is a block of polymethyl(methacrylate) (Perspex®)4 cm long (y dimension), 3 cm wide (x dimension) and 2 cm high (zdimension) in which a container 14 was hollowed to accommodate two cells12 a and 12 b, each for producing a specified proton concentration in anenvironment including an electrolyte, each cell 12 a or 12 b being 1 cmlong (y dimension), 0.6 cm wide (x dimension) and 1 cm deep (zdimension). Separating the hollows of container 14 corresponding to eachcell 12 a and 12 b is a 0.2 cm wide (x dimension) impermeable wall 64.Counter electrodes 24 a and 24 b of 0.1 mm thick platinum mesh wereplaced along a wall of cell 12 a and 12 b respectively. Workingelectrodes 26 a and 26 b were placed inside container 14, in parallel toand spaced 2 mm from counter electrodes 24 a and 24 b, protonconcentration sensors 32 a and 32 b (Orion 9863BN, Thermo FisherScientific Inc., Waltham, Mass., USA) were placed on the walls of cells12 a and 12 b opposite counter electrodes 24 a and 24 b, inside volumes30 a and 30 b.

Container 14 was filled with an electrolyte solution of 0.1 M Na₂SO₄ inwater. Each proton concentration sensor 32 a and 32 b was connected to asuitable display device to indicate what pH was measured by that sensorin a corresponding volume 30 a or 30 b.

Device 62 was used to produce a specified proton concentrationtopography in the electrolyte solution in a portion of container 14including volume 30 a, 30 b and the interface volume 74 there between.

Two electrical circuits were established, each circuit including anelectrode pair 24 a/26 a or 24 b/26 b and a separate independentvariable power supply. Each variable power supply was used toindependently pass a current of between about 0 and about 1 mA cm⁻² at apotential of between about 0 and about 5V through a respectiveelectrical circuit. The current passing through the circuit led tohydrolysis of water and the generation of protons and hydroxyl anions inthe volume of electrolyte chamber 28 a or 28 b. The concentration ofprotons increased in proximity of the cathode while the concentration ofprotons decreased in proximity of the anode. Since working electrodes 26a and 26 b were of mesh and therefore permeable to ions, generated ionspassed from the vicinity of a working electrode 26 through the workingelectrodes 26 into the electrolyte solution held in a respectiveassociated volume 30 a or 30 b.

The current applied between counter electrode 24 a and working electrode26 a was adjusted with reference to the readings of proton concentrationsensor 32 a until a proton concentration corresponding to a pH of 5.2was measured in the electrolyte solution held in volume 30 a associatedwith working electrode 26 a while the current applied between counterelectrode 24 b and working electrode 26 b was adjusted with reference tothe readings of proton concentration sensor 32 b until a protonconcentration corresponding to a pH of 9 was measured in theelectrolytic solution held in volume 30 b associated with workingelectrode 26 b, see FIG. 8C.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.

Section headings are used herein to ease understanding of thespecification and should not be construed as necessarily limiting.

What is claimed is:
 1. A method of producing a specified protonconcentration topography in an environment including an electrolyte,comprising: providing a plurality of independently controllable cells,each cell comprises a counter electrode, a working electrode, anelectrolysis volume between said counter electrode and a first side ofsaid working electrode, and a second volume on a second side of saidworking electrode opposite to said first side, each cell configured toproduce a specified proton concentration in said second volume; fillingsaid electrolysis volume and said second volume of each of saidplurality of cells with an environment including an electrolyte;specifying a desired proton concentration topography; and activatingeach said cell of said plurality of cells, so as to produce a specifiedproton concentration in each said second volume of said environmentwherein said specified proton concentrations generated in each saidsecond volume collectively constitute the specified proton concentrationtopography.
 2. The method of claim 1, further comprising: specifying adesired newly specified proton concentration topography different than apreviously defined proton concentration topography; and activating asaid cell of said plurality of cells, to produce a different protonconcentration in a said second volume of said environment therebychanging said proton concentration topography to be said newly specifiedproton concentration topography.
 3. The method of claim 1, furthercomprising: changing a proton concentration in at least one saidassociated second volume as a function of time, thereby changing saidproton concentration topography as a function of time.
 4. The method ofclaim 1, wherein movement of ions between two neighboring second volumesis substantially uninhibited.
 5. The method of claim 1, wherein saidactivating comprises independently varying a magnitude of each of aplurality of individually controllable electrical currents to producesaid desired proton concentration topography, each one of said pluralityof individually controllable electrical currents is passed between eachsaid working electrode and each said counter electrode.
 6. The method ofclaim 5, wherein each said working electrode and each said cell of saidplurality of independently controllable cells are between said counterelectrode and said environment.
 7. The method of claim 5, wherein eachsaid working electrode is permeable to a passage of electrolysisproducts therethrough.
 8. The method of claim 1, further comprising:monitoring said proton concentration in each said second volume; andadjusting a current passing between a respective working electrodelocated in a respective said independently controllable cell and arespective counter electrode so as to maintain said protonconcentration.
 9. The method of claim 5, further comprising: changingsaid plurality of individually controllable electrical currents as afunction of time, thereby changing said specified proton concentrationin each said second volume of said environment as a function of time.